Application protocol

Rapid antigen-specific T cell enrichment (Rapid ARTE)

Direct ex vivo characterization of human antigen-specific CD154+CD4+ T cells  

 CD154 is transiently up-regulated on activated CD4+ T cells and plays an important role as a co-stimulatory molecule in T cell/ag-presenting cell interactions through ligation of CD40. Due to its transient expression within hours after activation, CD154 can be used as a marker for activated ag-specific CD4+ T cells. Adding a CD40-blocking antibody during the stimulation of cell suspensions prevents down-regulation of CD154 expression induced by the interaction with CD40 expressed on ag-presenting cells. Combining magnetic cell enrichment and multiparameter flow cytometry analysis of CD154+CD4+ T cells allows the direct ex vivo detection and characterization of rare ag-specific T cells.

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Materials

The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.

Generation of PBMCs and in vitro stimulation for induction of CD154 expression

PBMC cultivation

  • RPMI 1640 medium
  • 100× L-glutamine stock solution (200 mM)
  • Human AB serum
  • 24-well plate (e.g., Gas-permeable Culture Plate # 150-000-362)
    ▲ Note: With the Gas-permeable Culture Plate # 150-000-362, up to 2.5×107 PBMCs/well/mL can be stimulated as opposed to 1×107 PBMCs/well/mL in standard 24-well plates.

PBMC stimulation

  •  CD40 pure – functional grade, human (# 130-094-133)
  • Reagents for  ag-specific T cell stimulation (e.g., PepTivator BKV VP1 – research grade, human, # 130-097-272 and PepTivator BKV LT – research grade, human, # 130-096-504)
  • Brefeldin A
  • FcR Blocking Reagent, human (# 130-059-901)

Magnetic and immunofluorescent surface labeling

Buffer (standard wash and dilution buffer)

  • autoMACS® Rinsing Solution (# 130-091- 222)
  • Bovine serum albumin (MACS® BSA Stock Solution; # 130-091-376)
  • CD154 MicroBead Kit, human (# 130-092-658)
  • Inside Stain Kit (# 130-090-477)
  • Orbital shaker
  • CD3-VioBlue®, human (#130-094-363)
  • CD4-APC, human (# 130-092-374)
  • CD8-PerCP, human (# 130-094-972)
  • CD14-PerCP, human (# 130-094-969)
  •  CD20-PerCP, human (# 130-094-976)

Magnetic enrichment and intracellular staining

  • MS Columns (# 130-042-201)
  • MACS Separator for MS columns (e.g. MiniMACS™ Separator # 130-042-102)
  • MACS Multistand  (#130-042-303)
  • CD154-FITC (# 130-096-233)
  • (Optional) Anti-cytokine antibodies for intracellular staining (e.g., Anti-IFN-g-PE # 130-091-653 or Anti-IL-17A-PE # 130-094-521)
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Protocol


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This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step.

Things to prepare in advance

PBMC medium

  • 500 mL RPMI1640
    + 5 mL 100× L-glutamine stock solution (2 mM final concentration)
    + 25 mL human AB serum (5% final concentration)

Reconstitution of PepTivators

  1. For reconstitution of the lyophilized peptide pool take the vial from –20 °C and warm-up to room temperature.
    ▲ Note: Do not open the vial by removing the rubber plug.
  2. To dissolve the 6 nmol version of the PepTivator, fill a sterile syringe (0.5 mL) with 200 μL of sterile water. To dissolve the 60 nmol version of the PepTivator, fill a sterile syringe (5 mL) with 2 mL of sterile water.
  3. Slowly inject the water with a sterile needle through the center of the rubber plug into the vial containing the lyophilized peptide pool.
  4. Vortex the solution to completely dissolve the lyophilized peptide pool. The concentration of the stock solution of PepTivator Peptides is 30 nmol (approximately 50 μg) of each peptide per mL. 
  5. Remove the rubber plug and aspirate the stock solution with a pipette.
  6. To avoid repeated freeze-thaw cycles, prepare working aliquots from the stock solution.
  7. Store the working aliquots at –80 °C. 

Generation of PBMCs

  1. Perform a sterile preparation of PBMCs from fresh buffy coats or whole blood using Ficoll-Paque™.
    ▲ Note: The following may be a useful protocol:
    Isolation of mononuclear cells from human peripheral blood by density gradient centrifugation
    ▲ Note: To remove platelets after density gradient separation, resuspend cell pellet in buffer and centrifuge at 200×g for 10−15 minutes at 20 °C. Carefully aspirate supernatant. Repeat washing step.
  2. Proceed to "in vitro stimulation for induction of CD154 expression".

In vitro stimulation for induction of CD154 expression

  1. Resuspend cells at a density of 1×107 cells/mL in culture medium RPMI 1640 with L-glutamine and 5% human AB serum.
  2. For each non-stimulated control and antigen-stimulated sample transfer 1×107 cells to one well of a 24-well plate.
    ▲ Note: With the Gas-permeable Culture Plate # 150-000-362, up to 2.5×107 PBMCs/well/mL can be stimulated as opposed to 1×107 PBMCs/well/mL in standard 24-well plates.
  3. Add 10 μL of CD40 pure – functional grade (1 μg/mL; 1:100) to each well.
  4. Add antigen according to manufacturer’s recommendation to the antigen-stimulated wells, but not to control wells. When using PepTivators: Add 20µl of reconstituted stock solution per well.
  5. Incubate cells for 4 hours at 37 °C, 5% CO2.
  6. Add brefeldin A to a final concentration of 2 µg/mL.
  7. Incubate cells for another 2 hours at 37 °C, 5% CO2.

Things to prepare in advance

Buffer (standard wash and dilution buffer)

  • Prepare a solution of PBS, pH 7.2, 2 mM EDTA and 0.5% BSA by diluting MACS® BSA Stock Solution 1:20 with autoMACS® Rinsing Solution. 

Antibody staining mix

Freshly prepare 200 μL of antibody staining mix for each well:

  • 12.5 μL CD3-VioBlue®
  • 25 μL CD4-APC
  • 25 μL CD8-PerCP
  • 25 μL CD14-PerCP
  • 50 μL CD20-PerCP
  • 25 μL CD154-Biotin(CD154 MicroBead Kit)
  • 37.5 μL FcR Blocking Reagent

Magnetic and immunofluorescent surface labeling

  1. Centrifuge plate for 5 minutes at 300×g at room temperature.
  2. Very carefully aspirate 800 μL of cell culture supernatant. Avoid resuspension of the cells.
  3. Add 200 μL of antibody staining mix to each well (see above)
  4. Mix well for 2 minutes using an orbital shaker.
  5. Incubate for 3 minutes in the dark at room temperature.
  6. Add 20 μL of Anti-Biotin MicroBeads UltraPure (from CD154 MicroBead Kit) to each well. Mix well for 2 minutes using an orbital shaker.
  7. Incubate for 13 minutes in the dark at room temperature.
  8. Add 300 μL of Inside Fix (from Inside Stain Kit) to each well. Mix well for 2 minutes using an orbital shaker.
  9. Incubate for 13 minutes in the dark at room temperature.
  10. Add 1 mL of buffer to each well and mix well.
  11. (Optional) Take an aliquot of 200 μL cell suspension for staining of the original fraction.

Magnetic enrichment and intracellular staining (on column) 

  1. Place MS Column in the magnetic field of a suitable MACS® Separator.
  2. Prepare MS Column by rinsing with 500 μL of buffer.
  3. Apply cell suspension onto the column.
  4. Wash cells by rinsing the column with 500 μL of buffer, followed by 2×500 μL of Inside Perm (from Inside Stain Kit).
  5. Prepare a solution of 10 μL of CD154-FITC and 90 μL of Inside Perm (from Inside Stain Kit).
  6. (Optional) Add additional staining antibodies to the solution, e.g., for the staining of cell surface antigens internalized upon cell activation or antigens which accumulate in the cell.
    ▲ Note: Do not exceed the total volume of 150 μL.
  7. Apply the solution onto the column and incubate for 10 minutes at room temperature.
    ▲ Note: The MACS Column has a flow-stop mechanism that will retain the solution in the column.
  8. Wash cells by rinsing the column with 2×500 μL of Inside Perm (from Inside Stain Kit) followed by 500 μL of buffer.
  9. Remove column from the separator and place it on a suitable collection tube.
  10. Pipette 500 μL of buffer onto the column.
  11. Cells are now ready for analysis. Store cells at 2–8 °C in the dark until analysis. Mix well before flow cytometry acquisition.
    ▲ Note: Samples may be stored at 2–8 °C in the dark for up to 24 hours.
    ▲ Note: Do not use propidium iodide (PI) or 7-AAD staining in general.

Intracellular staining of original samples

  1. Wash cells by adding 1 mL of buffer. Centrifuge cells for 5 minutes at 300×g at room temperature.
  2.  Aspirate supernatant.
  3. Resuspend cells in 500 μL of Inside Perm (from Inside Stain Kit) vortex well. Centrifuge cells for 5 minutes at 300×g.
  4. Aspirate supernatant.
  5. Prepare a solution of 10 μL of CD154-FITC and 90 μL of Inside Perm (from Inside Stain Kit).
  6. (Optional) Add additional staining antibodies to the solution, e.g., for the staining of cell surface antigens internalized upon cell activation or antigens which accumulate in the cell.
    ▲ Note: Do not exceed the total volume of 150 μL.
  7. Add staining solution to the cells and incubate for 10 minutes at room temperature.
  8. Wash cells by adding 1 mL of Inside Perm (from Inside Stain Kit). Centrifuge cells for 5 minutes at 300×g.
  9. Aspirate supernatant.
  10. Resuspend cells in 250 μL of buffer.
  11. Samples are ready for measurement now.
    ▲ Note: Samples may be stored at 2–8 °C in the dark for up to 24 hours.
View details

BKV-specific T cells detection without the Rapid ARTE protocol. (A) PBMC from six randomly selected healthy donors were stimulated with various peptide pools for immunodominant antigens of BKV, JCV, CMV, influenza, EBV, and AdV, and for positive control with a mixture of CMV/EBV/influenza MHC class I–restricted peptides. After 6 hours, IFN-g production within the CD4+ and CD8+ T cell compartments was analyzed by intracellular staining using the Rapid Cytokine Inspector. CMV-, EBV-, and AdV-specific IFN-γ+ T cells were clearly detectable in several donor samples. (B) In contrast, IFN-γ+ BKV and JCV specific T cells were detectable only at very low frequencies, between 0.01 and 0.03%, in five out of six donor samples. 

View details

The Rapid ARTE protocol successfully enriches ag-specific T cells. In blood samples BKV-specific T cells are present only at very low frequencies, in the range of the detection limit of flow cytometry analysis. The Rapid ARTE protocol enables enumeration of BKV-specific T cells. Up to 2.5×107 PBMC from 14 healthy donors were left untreated or stimulated with BKV peptide pools covering the complete sequence of the large T antigen (LT) and virion protein VP1. Both proteins are immunodominant  targets for T cell immunity. After six hours, cells were treated as described in this protocol, and the absolute numbers of enriched CD154+CD4+ T cells were determined. In blood samples of each donor, BKV-reactive CD4+ T cells were found. Absolute numbers ranged between 87 and 2340 per 1×107 PBMC.

View details

The Rapid ARTE approach allows processing larger cell numbers compared to detection by flow cytometry alone. (A) The protocol enabled the reliable characterization of functional subsets of the entire BKV-reactive CD154+CD4+ T cell pool, i.e., TNF-α-, IFN-g-, IL-10-, and IL-2-producing T cells. PBMC (2.5 × 107) from six healthy donors were processed as described in in this protocol. (B) Subsequently, the cytokine profile of the BKV-reactive CD4+ T cells was analyzed. TNF-α and IL-2 were expressed by the majority of BKV-specific T cells in all samples analyzed for these cytokines. In contrast, the frequency of IFN-γ+ cells among BKV-reactive CD4+ T cells varied between 27 and 74% in the different donor samples. Furthermore, IL-10-producing T cells could be identified at very low frequencies, which could not have been detected by flow cytometry without pre-enrichment.

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